A 22-kDa protein, caveolin, is localized to the cytoplasmic surface of plasma membrane specializations called caveolae. We have proposed that caveolin may function as a scaffolding protein to organize and concentrate signaling molecules within caveolae. Here, we show that caveolin interacts with itself to form homooligomers. Electron microscopic visualization ofthese purified caveolin homooligomers demonstrates that they appear as individual spherical particles. By using recombinant expression of caveolin as a glutathione S-transferase fusion protein, we have defined a region of caveolin's cytoplasmic N-terminal domain that mediates these caveolin-caveolin interactions. We suggest that caveolin homooligomers may function to concentrate caveolin-interacting molecules within caveolae. In this regard, it may be useful to think of caveolin homooligomers as fTishing lures" with multiple "hooks" or attachment sites for caveolin-interacting molecules.Caveolae are plasma membrane specializations (1). Caveolin, a 21-to 24-kDa integral membrane protein, has been identified as a principal component of caveolae membranes in vivo (2, 3). Purification of caveolin-rich membrane domains reveals several distinct classes of signaling molecules (3-6). These include heterotrimeric guanine nucleotide binding proteins (G proteins; a and 13'y subunits), Src-like kinases, protein kinase Ca, and Rap GTPases. Based on these observations, we have proposed (1) that caveolin may function as a scaffolding protein to organize and concentrate inactive signaling molecules within caveolae membranes-for activation by appropriate receptors. This caveolae signaling hypothesis states that "compartmentalization of ceftain cytoplasmic signaling molecules within caveolae could allow efficient and rapid coupling of activated receptors to more than one effector system" (1). In support of this view, inactive G a subunits interact directly with caveolin in a 1:1 stoichiometry-holding them in an inactive conformation (7). Thus, knowledge of the subunit structure of caveolin is important for understanding how caveolin might function to organize or concentrate G a subunits and other signaling molecules within caveolae membranes.In this report, we show that caveolin interacts with itself to form a discrete high molecular mass oligomer. As caveolin also interacts with G a subunits, self oligomerization of caveolin could provide a means for concentrating trimeric G proteins and other caveolin-interacting molecules within caveolae. The existence of caveolin homooligomers complexed with inactive G proteins could explain the observations of Rodbell and colleagues (8), who observed that inactive G proteins exist as high molecular mass oligomeric complexes and that activated G proteins dissociate to monomers. Similarly, activated G proteins fail to interact with recombinant caveolin (7). MATERIALS & METHODSMaterials. Antibodies to carbonic anhydrase IV and glutathione S-transferase (GST) were gifts of W. S. Sly (St. Louis University) and R. Young (Whitehead I...

MATERIALS AND METHODSPreparation of Thymosins. (3 and thymosin a, (a,), provided by Alpha 1 Biomedicals (Washington), were synthesized as described (25,26). The concentration of 4 was determined by the bicinchoninic acid assay (BCA; Sigma) with bovine serum albumin as the reference and was calibrated with amino acid analysis of (4.Actin Polymerization Assay. The activity of synthetic P4 in vitro was determined by the pyrene-conjugated actin assay. Pyrene-actin was prepared as described by Kouyama and Mihashi (27)

Abstract. The acrosomal process of Limulus sperm is an 80-~m long finger of membrane supported by a crystalline bundle of actin filaments. The filaments in this bundle are crosslinked by a 102-kD protein, scruin present in a 1:1 molar ratio with actin. Recent image reconstruction of scruin decorated actin illaments at 13-/~ resolution shows that scruin is organized into tv~ equally sized domains bound to separate actin subunits in the same filament. We have cloned and sequenced the gene for scruin from a Limulus testes cDNA library. The deduced amino acid sequence of scruin reflects the domain organiTation of scruin: it consists of a tandem pair of homologous domains joined by a linker region. The domain organization of scruin is confirmed by limited proteolysis of the purified acrosomal process. Three different proteases cleave the native protein in a 5-kD Proteasesensitive region in the middle of the molecule to generate an NH2-terminal 47-kD and a COOHterminal 56-kD protease-resistant domains. Although the protein sequence of scruin has no homology to any known actin-binding protein, it has similarities to several proteins, including four open reading frames of unknown function in poxviruses, as well as kelch, a Drosophila protein localized to actin-rich ring canals. All proteins that show homologies to scruin are characterized by the presence of an ,,~50-amino acid residue motif that is repeated between two and seven times. Crystallographic studies reveal this motif represents a four/~-stranded fold that is characteristic of the "superbarrel" structural fold found in the sialidase family of proteins. These results suggest that the two domains of scruin seen in EM reconstructions are superbarrel folds, and they present the possibility that other members of this family may also bind actin.

During activation of the Limulus sperm acrosomal process, actin filaments undergo a change in twist that is linked with the conversion from a coiled to a straight scruin-actin bundle. Since scruin had not been purified, its identity as an actin-binding protein has not been demonstrated. Using HECAMEG (methyl-6-O-(N-heptylcarbamoyl)-␣-D-glucopyranoside) detergent extraction in concert with high calcium, we purified native scruin and identified it as an equimolar complex with calmodulin. 125 I-Calmodulin overlays and calmodulin-Sepharose indicate that scruin binds calmodulin in calcium but not in EGTA. Overlay experiments also map the calmodulin binding site between the putative N-and C-terminal ␤-propeller domains within residues 425-446. Immunofluorescence microscopy reveals that calmodulin colocalizes with scruin and actin in the coiled bundle. Although scruin binds calmodulin, pelleting assays and electron microscopy show that the scruin cross-links F-actin into bundles independently of calcium. Based on our biochemical and structural studies, we suggest a model to explain how scruin controls a change in twist of actin filaments during the acrosome reaction. We predict that calcium subtly alters scruin conformation through its calmodulin subunit and the conformation change in scruin causes a shift in the relative positions of the scruin-bound actin subunits.